The growth of new blood vessels from existing endothelium (angiogenesis) is tightly controlled in healthy adults by opposing effects of positive and negative regulations. Under certain pathological conditions, including proliferative retinopathies, rheumatiod arthritis, psoriasis and cancer, positive regulations prevail and angiogenesis contributes to disease progression (Folkman (1995) Nature Medicine 1:27–31; Achen and Stacker (1998) Int. J. Exp. Pathol. 79:255–265). In cancer, the notion that angiogenesis represents the rate limiting step of tumor growth and metastasis, (Folkman (1971) New Engl. J. Med. 285:1182–1186) is now supported by considerable experimental evidence (reviewed in Aznavoorian et al. (1993) Cancer 71:1368–1383; Bidfer and Ellis (1994) Cell 79:185–188; Plate and Warnke (1997) J. Neurooncol 35:365–372; de Jong et al. (1998) J. Pathol 184:44–52).
A number of angiogenic growth factors have been described to date among which vascular endothelial growth factor (VEGF) appears to play a key role in the regulation of vasculogenesis and angiogenesis as a highly specific mitogen for endothelial cells (Brown et al., (1997) Control of Angiogenesis (Goldberg and Rosen, eds) Birkhauser, Basel, pp 233–269; Martiny-Broun and Marme(1995) Current Opin. in Biotech. 6:675–680; Ferrara and Davis-Smyth (1997) Endocrine Reviews 18:4–25).
VEGF is a glycosylated, disulfide-linked homodimeric protein consisting of two 23 kD subunits. Four different monomeric isoforms of VEGF exist ranging in size from 121 to 206 residues in humans (VEGF121, VEGF165, VEGF189 and VEGF206). Transcripts encoding the three shorter forms are detected in the majority of tumor cells and tumor tissue expressing VEGF gene. The isoforms result from different splicing events, and all variants share the same 115 N-terminal as well as six C-terminal residues and have a leader sequence to leave the cells. VEGF165 is the dominant isoform, while VEGF206 has so far only been identified in human fetal liver cDNA library VEGF165 and VEGF189 bind heparin with high affinity, and are sequestered to the cell surface or within the extracellular matrix bound to proteoglycans, while VEGF121 does not bind heparin and is thus freely diffusible. Plasmin cleavage of VEGF165 generates a 110-residue long N-terminal fragment (the receptor-binding domain) that no longer binds heparin but is equipotent to VEGF121 in its ability to induce endothelial cell proliferation.
VEGF is expressed in embryonic tissues (Breier et al., (1992) Development (Camb.) 114:521), macrophages, proliferating epidermal keratinocytes during wound healing (Brown et al., (1992) J. Ex. Med. 176:1375–9) and may be responsible for tissue edema associated with inflammation (Ferrara and Davis-Smyth (1997) Endocrine Reviews 18:4–25). In situ hybridization studies have demonstrated high VEGF expression in a number of human tumors including glioblastoma, ovarian tumors, carcinoma, hemangioblastoma, brain neoplasms and Kaposi's sarcoma (Plate et al., (1992) Nature 359:845–848; Zebrowski et al., (1999) Ann. Surg. Oncol. 6:373–378). High levels of VEGF were also observed in hypoxia-induced angiogenesis (Shweiki et al., (1992) Nature 359:843–845).
The biological function of VEGF is mediated through binding to two high affinity receptors which are selectively expressed on endothelial cells during embryogenesis (Millauer et al., (1993) Cell 72:835–838) and VEGF related pathologies (tumor formation). VEGF receptors include the human kinase domain receptor (KDR), described in U.S. Pat. No. 5,712,380; its murine analog flk-1, sequenced by Mallhews (1991) Proc. Natl. Acad. Sci. USA, 88:9026–9030; U.S. Pat. No. 5,270,458 and the Fsm-like tyrosine kinase (Flt-1) (Shibuya et al., (1990) Oncogene 5:519–524). All of them are class III tyrosine kinases (Vaisman et al., 1990: J. Biol. Chem. 265, 19461–19466; Kaipainen et al., (1993) J. Exp. Med. 178:2077–2088). Studies in mice have shown that the expression of KDR reaches the highest levels during embryonic vasculogenesis and angiogenesis (Millauer et al., 1993 Cell 72:835–838). In contrast, only low levels of mRNA for Flt-1 were found during fetal growth and moderate levels during organogenesis, but high levels in newborn mice (Peters et al., 1993 Proc. Natl. Aca. Sci. U.S.A 90(16):7533–7). Experiments with knockout mice deficient in either receptor revealed that KDR is essential for the development of endothelial cells, whereas Flt-1 is necessary for the organization of embryonic vasculature (Fong et al., 1995 Dev. Dyn. 203(1):80–92; Shalaby et al., 1995 Nature 376 (6535:62–6).
KDR and Flt-1, each ˜1300 amino acid residues long, are composed of 7 extracellular Ig-like domains containing the ligand-binding region, a single short membrane-spanning sequence, and an intracellular region containing tyrosine kinase domains. The amino acid sequences of KDR and Flt-1 are ˜45% identical to each other. Flt-1 has the higher affinity for VEGF (KD=10–20 pM) compared to 75–125 pM for the KDR receptor. VEGF binding to KDR but not Flt-1 elicits an efficient (ED50˜0.1–1 ng/ml) DNA synthetic and chemotactic endothelial cell response. Activation of Flt-1 receptor by VEGF might modulate the interaction of endothelial cells with each other or the basement membrane on which they reside.
The Flt-1 receptor mRNA can be spliced to generate forms encoding either the full-length membrane-spanning receptor or a soluble form, denoted sFlt-1. Pure sFlt-1 retains its specific high affinity binding for VEGF and fully inhibits VEGF-stimulated endothelial cell mitogenesis by dominant negative mechanism.
Like other growth factor transmembrane tyrosine kinase receptors, VEGF receptors presumably undergo ligand-induced dimerization, that triggers signal transduction by promoting either autophosphorylation or transphosphorylation specific downstream signal transduction protein mediators.
To gain a better understanding of the biological activity of VEGF the analysis of structure/activity relationships was performed using site-directed mutagenesis and epitope mapping of neutralizing monoclonal antibodies (Keyt et al., (1996) J. Biol. Chem. 271:5638–5646). Arg82, Lys84 and His86, located in a hairpin loop, were found to be critical for binding KDR/Flk-1, while negatively charged residues, Asp63, Glu64 and Glu67, were associated with Flt-1 binding. The three-dimensional structure of the receptor-binding domain of VEGF (residues 8–109) showed that these positively and negatively charged regions are distal in the monomer but are spatially close in the dimer (Wiesmann et al., (1997) Cell 91:695–704). Mutations within the KDR site had minimal effect on Fit-1 binding, suggesting that receptors have different binding sites on VEGF which may serve to dimerize tyrosine kinase receptors resulting in initiation of angiogenesis.
Domain deletion studies on Flt-1 receptor have shown that the ligand binding function resides within the first three domains (Barleon et al., (1997) J. Biol. Chem. 272:10382–1038; Cunningham et al., (1997) Biochim. Biophys. Res. Commun. 231 (3): 596–599), and domain 4 is required to efficiently couple ligand binding to signal transduction by means of direct receptor-receptor contacts (Barleon et al., (1997) J. Biol. Chem. 272:10382–10388). The crystal structure of the complex between VEGF and the second domain of Flt-1 showed domain 2 in a predominantly hydrophobic interaction with the “poles” of VEGF dimer (Wiesmann et al., (1997) Cell 91:695–704). Deletion experiments on KDR demonstrated that only domain 2 and 3 are critical for ligand binding (Fuh et al., (1998) J. Biol Chem. 1998; 273 (18):11197–204).
Endothelial cells also contain another type of VEGF receptors, Neuropilins (NP), possessing a lower mass than either VEGFR2 or VEGFR1 (Gluzman-Poltorak Z., et al., (2000) J. Biol. Chem., 275(24):18040–5; WO Patent 0002/3565A2). It was subsequently found that these smaller VEGF receptors of endothelial cells are isoform specific receptors that bind VEGF165 but not VEGF121 (Gluzman-Poltorak Z, et al., (2000) J. Biol. Chem., 275(38):29922). Unusually large amounts of these isoform-specific receptors were found in several types of prostate and breast cancer cell lines (Miao, H. Q., et al., (2000) FASEB J., 14(15):2532–9). Neuropilin-1 is likely to play an important role in the development of the cardiovascular system. Gene disruption studies have indicated that np-1 participates in embryonic vasculogenesis and angiogenesis and is involved in the maturation of blood vessels, since mouse embryos lacking a functional np-1 gene die because their cardiovascular system fails to develop properly (Kawasaki, T., et al., (1999) Neurobiol. 39(4):579–89). Subsequent experiments have shown that NP-1 also serves as a receptor for the heparin-binding form of placenta growth factor (PlGF), PlGF-2, and for VEGF-B.
In addition to its normal physiological role, VEGF receptors are associated with numerous pathologies, including cancer, rheumatiod arthritis, diabetic retinopathy and psoriasis; development of VEGF antagonists, blocking the interaction between VEGF and its receptors with is therefore clinically attractive. Humanized neutralizing antibodies have been shown to interact with VEGF near the KDR and Flt-i binding sites (Kim, K. J., et al., (1993) Nature 362, 841–844; Muller, Y. A., et al., (1997) Proc. Nat. Acad. Sci. 94, 7192–7197; Muller, Y. A. et al., (1998) Structure 6:1153–1167; U.S. Pat. No. 5,855,866), and SELEX-derived RNA molecules (Jellinek, D. et al., (1994) Biochemistry 33:10450–10456; U.S. Pat. No. 5,859,228), that target VEGF, suppress tumor growth that is dependent on vascularization of adjacent normal tissue (Plate, K. H. et al., (1994) Brain-Pathol., 4:207–218). Anti KDR monoclonal antibodies inhibited VEGF induced signaling and demonstrated high anti-tumor activity (Witte et al., (1998) Cancer & Metast. Reviews 17:155–161; U.S. Pat. No. 5,840,301). Soluble Fit receptor (U.S. Pat. No. 5,861,484), fragments of VEGF (U.S. Pat. No. 5,240,848) have been shown to inhibit factor/receptor interaction and angiogenesis in vivo. Anti VEGF antisense oligonucleotide was designed to inhibit VEGF expression and VEGF induced neovascularization (U.S. Pat. No. 5,641,756).
The present invention describes a new ligand for VEGF receptor-1 and similar receptors with is useful for targeted delivery of therapeutics. Such treatment should be as devoid as possible of undesired side effects such as those associated with conventional chemotherapy and some of the experimental biotherapies.